Journal club: structure of the ternary Prp31:15.5K:U4 snRNA complex
Michael Durney
Monday, 09 April 2007 17:20 UTC
I would like to start the “journal club” section of the NMR forum by presenting my favorite paper from the recent literature.
The paper (in this weeks issue of Science) is, in my opinion at least, a perfect example of the ability of NMR to contribute to the structural analysis of a challenging RNA-protein complex
I chose this high-impact paper because the combination of biochemistry, NMR, and crystallography on a ternary complex is how I would like to tackle a challenging project that I am working on.
The authors used chemical shift perturbation and saturation transfer to monitor the effect of titrating Prp31 into the preformed U4-15.5K RNA-protein complex. To do this the authors purified nitrogen-15 labeled and deuterated samples of the 15.5K construct. All of the proteins were produced in E.coli using well-established protocols. Gel filtration was used to purify the complexes to yield homogeneous samples
Subsequent crystallogrpahic analysis confirmed formation of the ternary complex and provides a rationale for how Prp31 contributes to the stability of the complex. The structural studies in combination with biochemical experiments yield valuable insights into spliceosome mechanism. NMR experiments such as the ones reported here are well within the reach of most laboratories with access to instrumentation and can be very powerful in lending confidence to subsequent crystallization experiments.
Your comments are welcome!
Michael.
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Replies
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Really? Chemical shift perturbations and saturation transfer are what gets you excited? The entire NMR section of this paper was only a paragraph and they didn’t learn anything the crystal structure wouldn’t have told them. In terms of drug binding, the other work that Carlomango does with INPHARMA is incredibly exciting. Overall, I think this paper just boils down to another Science-worthy crystal structure, of which there are many, and doesn’t really do much service to the power of NMR.
Now, I don’t want it to sound like I’m bashing NMR, it’s one of the most powerful techniques available to get atomic resolution of dynamics and binding events. Chemical shift perturbations are indeed powerful, but don’t just use them to get more than a binding site (which can be difficult to distinguish from allosteric effects anyways), titrate the sample in and determine a Kd! If you’re lucky and you find yourself in slow exchange, do some ZZ-exchange experiments and get kon and koff rates. Same thing applies with the saturation transfer. Don’t just look for where, find out how! That’s the power of NMR. You can’t determine any of those things with the snapshots obtained from a crystal structure.
There really are some incredible NMR papers in high impact journals, though. I’ve included some of my more favorite references (of which there are many) below.
Visualizing spatially correlated dynamics that directs RNA conformational transitions
Zhang Q, Stelzer AC, Fisher CK, et al.
NATURE Volume: 450 Issue: 7173 Pages: 1263-U14Quantitative dynamics and binding studies of the 20S proteasome by NMR
Sprangers R, Kay LE
NATURE Volume: 445 Issue: 7128 Pages: 618-622Detecting transient intermediates in macromolecular binding by paramagnetic NMR
Iwahara J, Clore GM
NATURE Volume: 440 Issue: 7088 Pages: 1227-1230NMR analysis of a 900K GroEL-GroES complex
Fiaux J, Bertelsen EB, Horwich AL, et al.
NATURE Volume: 418 Issue: 6894 Pages: 207-211
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